Human osteoclast-specific and -related genes

ABSTRACT

The present invention relates to purified DNA sequences encoding all or a portion of an osteoclast-specific or -related gene products and a method for identifying such sequences. The invention also relates to antibodies directed against an osteoclast-specific or -related gene product. Also claimed are DNA constructs capable of replicating DNA encoding all or a portion of an osteoclast-specific or -related gene product, and DNA constructs capable of directing expression in a host cell of an osteoclast-specific or -related gene product.

RELATED APPLICATION

This application is a continuation of application Ser. No. 08/045,270 filed on Apr. 6, 1993 now abandoned.

BACKGROUND OF THE INVENTION

Excessive bone resorption by osteoclasts contributes to the pathology of many human diseases including arthritis, osteoporosis, periodontitis, and hypercalcemia of malignancy. During resorption, osteoclasts remove both the mineral and organic components of bone (Blair, H. C., et al., J. Cell Biol. 102:1164 (1986)). The mineral phase is solubilized by acidification of the sub-osteoclastic lacuna, thus allowing dissolution of hydroxyapatite (Vaes, G., Clin. Orthop. Relat. 231:239 (1988)). However, the mechanism(s) by which type I collagen, the major structural protein of bone, is degraded remains controversial. In addition, the regulation of osteoclastic activity is only partly understood. The lack of information concerning osteoclast function is due in part to the fact that these cells are extremely difficult to isolate as pure populations in large numbers. Furthermore, there are no osteoclastic cell lines available. An approach to studying osteoclast function that permits the identification of heretofore unknown osteoclast-specific or -related genes and gene products would allow identification of genes and gene products that are involved in the resorption of bone and in the regulation of osteoclastic activity. Therefore, identification of osteclast-specific or -related genes or gene products would prove useful in developing therapeutic strategies for the treatment of disorders involving aberrant bone resorption.

SUMMARY OF THE INVENTION

The present invention relates to isolated DNA sequences encoding all or a portion of osteoclast-specific or -related gene products. The present invention further relates to DNA constructs capable of replicating DNA encoding osteoclast-specific or -related gene products. In another embodiment, the invention relates to a DNA construct capable of directing expression of all or a portion of the osteoclast-specific or -related gene product in a host cell.

Also encompassed by the present invention are prokaryotic or eukaryotic cells transformed or transfected with a DNA construct encoding all or a portion of an osteoclast-specific or -related gene product. According to a particular embodiment, these cells are capable of replicating the DNA construct comprising the DNA encoding the osteoclast-specific or -related gene product, and, optionally, are capable of expressing the osteoclast-specific or -related gene product. Also claimed are antibodies raised against osteoclast-specific or -related gene products, or portions of these gene products.

The present invention further embraces a method of identifying osteoclast-specific or -related DNA sequences and DNA sequences identified in this manner. In one embodiment, cDNA encoding osteoclast is identified as follows: First, human giant cell tumor of the bone was used to 1) construct a cDNA library; 2) produce ³² P-labelled cDNA to use as a stromal cell⁺ ; osteoclast⁺ probe, and 3) produce (by culturing) a stromal cell population lacking osteoclasts. The presence of osteoclasts in the giant cell tumor was confirmed by histological staining for the osteoclast marker, type 5 tartrate-resistant acid phosphatase (TRAP) and with the use of monoclonal antibody reagents.

The stromal cell population lacking osteoclasts was produced by dissociating cells of a giant cell tumor, then growing and passaging the cells in tissue culture until the cell population was homogeneous and appeared fibroblastic. The cultured stromal cell population did not contain osteoclasts. The cultured stromal cells were then used to produce a stromal cell⁺, osteoclast⁻ ³² P-labelled cDNA probe.

The cDNA library produced from the giant cell tumor of the bone was then screened in duplicate for hybridization to the cDNA probes: one screen was performed with the giant cell tumor cDNA probe (stromal cell⁺, osteoclast⁺), while a duplicate screen was performed using the cultured stromal cell cDNA probe (stromal cell⁺, osteoclast⁻). Hybridization to a stromal⁺, osteoclast⁺ probe, accompanied by failure to hybridize to a stromal⁺, osteoclast⁻ probe indicated that a clone contained nucleic acid sequences specifically expressed by osteoclasts.

In another embodiment, genomic DNA encoding osteoclast -specific or -related gene products is identified through known hybridization techniques or amplification techniques. In one embodiment, the present invention relates to a method of identifying DNA encoding an osteoclast-specific or -related protein, or gene product, by screening a cDNA library or a genomic DNA library with a DNA probe comprising one or more sequences selected from the group consisting of the DNA sequences set out in Table I (SEQ ID NOs: 1-32). Finally, the present invention relates to an osteoclast-specific or related protein encoded by a nucleotide sequence comprising a DNA sequence selected from the group consisting of the sequences set out in Table I, or their complementary strands.

BRIEF DESCRIPTION OF FIG. 1

The FIG. 1 shows cDNA sequence (SEQ ID NO: 33) of human gelatinase B, and highlights those portions of the sequence represented by the osteoclast-specific or -related cDNA clones of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, Applicant has identified osteoclast-specific or osteoclast-related nucleic acid sequences. These sequences were identified as follows: Human giant cell tumor of the bone was used to 1) construct a cDNA library; 2) produce ³² P-labelled cDNA to use as a stromal cell⁺, osteoclast⁺ probe, and 3) produce (by culturing) a stromal cell population lacking osteoclasts. The presence of osteclasts in the giant cell tumor was confirmed by histological staining for the osteoclast marker, type 5 acid phosphatase (TRAP). In addition, monoclonal antibody reagents were used to characterize the multinucleated cells in the giant cell tumor, which cells were found to have a phenotype distinct from macrophages and consistent with osteoclasts.

The stromal cell population lacking osteoclasts was produced by dissociating cells of a giant cell tumor, then growing the cells in tissue culture for at least five passages. After five passages the cultured cell population was homogeneous and appeared fibroblastic. The cultured population contained no multinucleated cells at this point, tested negative for type 5 acid phosphatase, and tested variably alkaline phosphatase positive. That is, the cultured stromal cell population did not contain osteoclasts. The cultured stromal cells were then used to produce a stromal cell⁺, osteoclast⁻ ³² P-labelled cDNA probe.

The cDNA library produced from the giant cell tumor of the bone was then screened in duplicate for hybridization to the cDNA probes: one screen was performed with the giant cell tumor cDNA probe (stromal cell⁺, osteroclast⁺), while a duplicate screen was performed using the cultured stromal cell cDNA probe (stromal cell⁺ osteoclast⁻) Clones that hybridized to the giant cell tumor cDNA probe (stromal⁺, osteoclast⁺), but not to the stromal cell cDNA probe (stromal⁺, osteoclast⁻), were assumed to contain nucleic acid sequences specifically expressed by osteoclasts.

As a result of the differential screen described herein, DNA specifically expressed in osteoclast cells characterized as described herein was identified. This DNA, and equivalent DNA sequences, is referred to herein as osteoclast-specific or osteoclast-related DNA. Osteoclast-specific or -related DNA of the present invention can be obtained from sources in which it occurs in nature, can be produced recombinantly or synthesized chemically; it can be cDNA, genomic DNA, recombinantly-produced DNA or chemically-produced DNA. An equivalent DNA sequence is one which hybridizes, under standard hybridization conditions, to an osteoclast-specific or -related DNA identified as described herein or to a complement thereof.

Differential screening of a human osteoclastoma cDNA library was performed to identify genes specifically expressed in osteoclasts. Of 12,000 clones screened, 195 clones were identified which are either uniquely expressed in osteoclasts, or are osteoclast-related. These clones were further identified as osteoclast-specific, as evidenced by failure to hybridize to mRNA derived from a variety of unrelated human cell types, including epithelium, fibroblasts, lymphocytes, myelomonocytic cells, osteoblasts, and neuroblastoma cells. Of these, 32 clones contain novel cDNA sequences which were not found in the GenBank database.

A large number of cDNA clones obtained by this procedure were found to represent 92 kDa type IV collagenase (gelatinase B; E.C. 3.4.24.35) as well as tartrate resistant acid phosphatase. In situ hybridization localized mRNA for gelatinase B to multinucleated giant cells in human osteoclastomas. Gelatinase B immunoreactivity was demonstrated in giant cells from 8/8 osteoclastomas, osteoclasts in normal bone, and in osteoclasts of Paget's disease by use of a polyclonal antisera raised against a synthetic gelatinase B peptide. In contrast, no immunoreactivity for 72 kDa type IV collagenase (gelatinase A; E.C. 3.4.24.24), which is the product of a separate gene, was detected in osteoclastomas or normal osteoclasts.

The present invention has utility for the production and identification of nucleic acid probes useful for identifying osteoclast-specific or -related DNA. Osteoclast-specific or -related DNA of the present invention can be used to produce osteoclast-specific or -related gene products useful in the therapeutic treatment of disorders involving aberrant bone resorption. The osteoclast-specific or -related sequences are also useful for generating peptides which can then be used to produce antibodies useful for identifying osteoclast-specific or -related gene products, or for altering the activity of osteoclast-specific or -related gene products. Such antibodies are referred to as osteoclast-specific antibodies. Osteoclast-specific antibodies are also useful for identifying osteoclasts. Finally, osteoclast -specific or -related DNA sequences of the present invention are useful in gene therapy. For example, they can be used to alter the expression in osteoclasts of an aberrant osteoclast -specific or -related gene product or to correct aberrant expression of an osteoclast-specific or -related gene product. The sequences described herein can further be used to cause osteoclast-specific or related gene expression in cells in which such expression does not ordinarily occur, i.e., in cells which are not osteoclasts.

Example 1--Osteoclast cDNA Libary Construction

Messenger RNA (mRNA) obtained from a human osteoclastoma (`giant cell tumor of bone`), was used to construct an osteoclastoma cDNA library. Osteoclastomas are actively bone resorptive tumors, but are usually non-metastatic. In cryostat sections, osteoclastomas consist of ˜30% multinucleated cells positive for tartrate resistant acid phosphatase (TRAP), a widely utilized phenotypic marker specific in vivo for osteoclasts (Minkin, Calcif. Tissue Int. 34:285-290 (1982)). The remaining cells are uncharacterized `stromal` cells, a mixture of cell types with fibroblastic/mesenchymal morphology. Although it has not yet been definitively shown, it is generally held that the osteoclasts in these tumors are non-transformed, and are activated to resorb bone in vivo by substance(s) produced by the stromal cell element.

Monoclonal antibody reagents were used to partially characterize the surface phenotype of the multinucleated cells in the giant cell tumors of long bone. In frozen sections, all multinucleated cells expressed CD68, which has previously been reported to define an antigen specific for both osteoclasts and macrophages (Horton, M. A. and M. H. Helfrich, In Biology and Physiology of the Osteoclast, B. R. Rifkin and C. V. Gay, editors, CRC Press, Inc. Boca Raton, Fla., 33-54 (1992)). In contrast, no staining of giant cells was observed for CD11b or CD14 surface antigens, which are present on monocyte/macrophages and granulocytes (Arnaout, M. A. et al. J. Cell. Physiol. 137:305 (1988); Haziot, A. et al. J. Immunol. 141:547 (1988)). Cytocentrifuge preparations of human peripheral blood monocytes were positive for CD68, CD11b, and CD14. These results demonstrate that the multinucleated giant cells of osteoclastomas have a phenotype which is distinct from that of macrophages, and which is consistent with that of osteoclasts.

Osteoclastoma tissue was snap frozen in liquid nitrogen and used to prepare poly A⁺ mRA according to standard methods. cDNA cloning into a pcDNAII vector was carried out using a commercially-available kit (Librarian, InVitrogen). Approximately 2.6×10⁶ clones were obtained, >95% of which contained inserts of an average length 0.6 kB.

Example 2--Stromal Cell mRNA Preparation

A portion of each osteoclastoma was snap frozen in liquid nitrogen for mRNA preparation. The remainder of the tumor was dissociated using brief trypsinization and mechanical disaggregation, and placed into tissue culture. These cells were expanded in Dulbecco's MEM (high glucose, Sigma) supplemented with 10% newborn calf serum (MA Bioproducts), gentamycin (0.5 mg/ml), 1-glutamine (2 mM) and non-essential amino acids (0.1 mM) (Gibco). The stromal cell population was passaged at least five times, after which it showed a homogenous, fibroblastic looking cell population that contained no multinucleated cells. The stromal cells were mononuclear, tested negative acid phosphatase, and tested variably alkaline phosphatase positive. These findings indicate that propagated stromal cells (i.e., stromal cells that are passaged in culture) are non-osteoclastic and non-activated.

Example 3--Identification of DNA Encoding Osteoclastoma-Specific or -Related Gene Products by Differential screening of an Osteoclastoma cDNA Library

A total of 12,000 clones drawn from the osteoclastoma cDNA library were screened by differential hybridization, using mixed ³² P labelled cDNA probes derived from (1) giant cell tumor mRNA (stromal cell⁺, OC⁺), and (2) mRNA from stromal cells (stromal cell⁺, OC⁺) cultivated from the same tumor. The probes were labelled with ³² [P]dCTP by random priming to an activity of ˜10⁹ CPM/μg. Of these 12,000 clones, 195 gave a positive hybridization signal with giant cell (i.e., osteoclast and stromal cell) mRNA, but not with stromal cell mRNA. Additionally, these clones failed to hybridize to cDNA produced from mRNA derived from a variety of unrelated human cell types including epithelial cells, fibroblasts, lymphocytes, myelomonocytic cells, osteoblasts, and neuroblastoma cells. The failure of these clones to hybridize to cDNA produced from mRNA derived from other cell types supports the conclusion that these clones are either uniquely expressed in osteoclasts, or are osteoclast-related.

The osteoclast (OC) cDNA library was screened for differential hybridization to OC cDNA (stromal cell⁺, OC⁺) and stromal cell cDNA (stromal cell⁺, OC⁻) as follows:

NYTRAN filters (Schleicher & Schuell) were placed on agar plates containing growth medium and ampicillin. Individual bacterial colonies from the OC library were randomly picked and transferred, in triplicate, onto filters with preruled grids and then onto a master agar plate. Up to 200 colonies were inoculated onto a single 90-mm filter/plate using these techniques. The plates were inverted and incubated at 37° C. until the bacterial inoculates had grown (on the filter) to a diameter of 0.5-1.0 mm.

The colonies were then lysed, and the DNA bound to the filters by first placing the filters on top of two pieces of Whatman 3 MM paper saturated with 0.5N NaOH for 5 minutes. The filters were neutralized by placing on two pieces of Whatman 3 MM paper saturated with 1M Tris-HCL, pH 8.0 for 3-5 minutes. Neutralization was followed by incubation on another set of Whatman 3 MM papers saturated with 1M Tris-HCL, pH 8.0/1.5M NaCl for 3-5 minutes. The filters were then washed briefly in 2×SSC.

DNA was immobilized on the filters by baking the filters at 80° C. for 30 minutes. Filters were best used immediately, but they could be stored for up to one week in a vacuum jar at room temperature.

Filters were prehybridized in 5-8 ml of hybridization solution per filter, for 2-4 hours in a heat sealable bag. An additional 2 ml of solution was added for each additional filter added to the hybridization bag. The hybridization buffer consisted of 5×SSC, 5×Denhardt's solution, 1% SDS and 100 μg/ml denatured heterologous DNA.

Prior to hybridization, labeled probe was denatured by heating in 1×SSC for 5 minutes at 100° C., then immediately chilled on ice. Denatured probe was added to the filters in hybridization solution, and the filters hybridized with continuous agitation for 12-20 hours at 65° C.

After hybridization, the filters were washed in 2×SSC/0.2% SDS at 50°-60° C. for 30 minutes, followed by washing in 0.2×SSC/0.2% SDS at 60° C. for 60 minutes.

The filters were then air dried and autoradiographed using an intensifying screen at -70° C. overnight.

Example 4--DNA Sequencing of Selected Clones

Clones reactive with the mixed tumor probe, but unreactive with the stromal cell probe, are expected to contain either osteoclast-related, or in vivo `activated` stromal-cell-related gene products. One hundred and forty-four cDNA clones that hybridized to tumor cell cDNA, but not to stromal cell cDNA, were sequenced by the dideoxy chain termination method of Sanger et al. (Sanger F., et al. Proc. Natl. Acad. Sci. USA 74:5463 (1977)) using sequenase (US Biochemical). The DNASIS (Hitatchi) program was used to carry out sequence analysis and a homology search in the GenBank/EMBL database.

Fourteen of the 195 tumor⁺ stromal⁻ clones were identified as containing inserts with a sequence identical to the osteoclast marker, type 5 tartrate-resistant acid phosphatase (TRAP) (GenBank accession number J04430 M19534). The high representation of TRAP positive clones also indicates the effectiveness of the screening procedure in enriching for clones which contain osteoclast-specific or related cDNA sequences.

Interestingly, an even larger proportion of the tumor⁺ stromal⁻ clones (77/195; 39.5%) were identified as human gelatinase B (macrophage-derived gelatinase) (Wilhelm, S. M. J. Biol. Chem. 264:17213 (1989)), again indicating high expression of this enzyme by osteoclasts. Twenty-five of the gelatinase B clones were identified by dideoxy sequence analysis; all 25 showed 100% sequence homology to the published gelatinase B sequence (Genbank accession number J05070). The portions of the gelatinase B cDNA sequence covered by these clones is shown in the FIGURE (SEQ ID NO: 33). An additional 52 gelatinase B clones were identified by reactivity with a ³² P-labelled probe for gelatinase B.

Thirteen of the sequenced clones yielded no readable sequence. A DNASIS search of GenBank/EMBL databases revealed that, of the remaining 91 clones, 32 clones contain novel sequences which have not yet been reported in the databases or in the literature. These partial sequences are presented in Table I. Note that three of these sequences were repeats, indicating fairly frequent representation of mRNA related to this sequence. The repeat sequences are indicated by^(a), b superscripts (Clones 198B, 223B and 32C of Table I).

                                      TABLE I                                      __________________________________________________________________________     PARTIAL SEQUENCES OF 32 NOVEL OC-SPECIFIC OR -RELATED                          EXPRESSED GENES (cDNA CLONES)                                                  34A (SEQ ID NO: 1)                                                             1  GCAAATATCT                                                                               AAGTTTATTG                                                                               CTTGGATTTC                                                                               TAGTGAGAGC                                                                               TGTTGAATTT                                                                               GGTGATGTCA                61 AATGTTTCTA                                                                               GGGTTTTTTT                                                                               AGTTTGTTTT                                                                               TATTGAAAAA                                                                               TTTAATTATT                                                                               TATGCTATAG                121                                                                               GTGATATTCT                                                                               CTTTGAATAA                                                                               ACCTATAATA                                                                               GAAAATAGCA                                                                               GCAGACAACA                          4B (SEQ ID NO: 2)                                                              1  GTGTCAACCT                                                                               GCATATCCTA                                                                               AAAATGTCAA                                                                               AATGCTGCAT                                                                               CTGGTTAATG                                                                               TCGGGGTAGG                61 GGG                                                                         12B (SEQ ID NO: 3)                                                             1  CTTCCCTCTC                                                                               TTGCTTCCCT                                                                               TTCCCAAGCA                                                                               GAGGTGCTCA                                                                               CTCCATGGCC                                                                               ACCGCCACCA                61 CAGGCCCACA                                                                               GGGAGTACTG                                                                               CCAGACTACT                                                                               GCTGATGTTC                                                                               TCTTAAGGCC                                                                               CAGGGAGTCT                121                                                                               CAACCAGCTG                                                                               GTGGTGAATG                                                                               CTGCCTGGCA                                                                               CGGGACCCCC                                                                               CCC                                 28B (SEQ ID NO: 4)                                                             1  TTTTATTTGT                                                                               AAATATATGT                                                                               ATTACATCCC                                                                               TAGAAAAAGA                                                                               ATCCCAGGAT                                                                               TTTCCCTCCT                61 GTGTGTTTTC                                                                               GTCTTGCTTC                                                                               TTCATGGTCC                                                                               ATGATGCCAG                                                                               CTGAGGTTGT                                                                               CAGTACAATG                121                                                                               AAACCAAACT                                                                               GGCGGGATGG                                                                               AAGCAGATTA                                                                               TTCTGCCATT                                                                               TTTCCAGGTC                                                                               TTT                       37B (SEQ ID NO: 5)                                                             1  GGCTGGACAT                                                                               GGGTGCCCTC                                                                               CACGTCCCTC                                                                               ATATCCCCAG                                                                               GCACACTCTG                                                                               GCCTCAGGTT                61 TTGCCCTGGC                                                                               CATGTCATCT                                                                               ACCTGGAGTG                                                                               GGCCCTCCCCC                                                                              TTCTTCAGCC                                                                               TTGAATCAAA                121                                                                               AGCCACTTTG                                                                               TTAGGCGAGG                                                                               ATTTCCCAGA                                                                               CCACTCATCA                                                                               CATTAAAAAA                                                                               TATTTTGAAA                181                                                                               ACAAAAAAAA                                                                               AAAAAAA                                                           55B (SEQ ID NO: 6)                                                             1  TTGACAAAGC                                                                               TGTTTATTTC                                                                               CACCAATAAA                                                                               TAGTATATGG                                                                               TGATTGGGGT                                                                               TTCTATTTAT                61 AAGAGTAGTG                                                                               GCTATTATAT                                                                               GGGGTATCAT                                                                               GTTGATGCTC                                                                               ATAAATAGTT                                                                               CATATCTACT                121                                                                               TAATTTGCCT                                                                               TC                                                                60B (SEQ ID NO: 7)                                                             1  GAAGAGAGTT                                                                               GTATGTACAA                                                                               CCCCAACAGG                                                                               CAAGGCAGCT                                                                               AAATGCAGAG                                                                               GGTACAGAGA                61 GATCCCGAGG                                                                               GAATT                                                             86B (SEQ ID NO: 8)                                                             1  GGATGGAAAC                                                                               ATGTAGAAGT                                                                               CCAGAGAAAA                                                                               ACAATTTTAA                                                                               AAAAAGGTGG                                                                               AAAAGTTACG                61 GCAAACCTGA                                                                               GATTTCAGCA                                                                               TAAAATCTTT                                                                               AGTTAGAAGT                                                                               GAGAGAAAGA                                                                               AGAGGGAGGC                121                                                                               TGGTTGCTGT                                                                               TGCACGTATC                                                                               AATAGGTTAT                                                                               C                                             87B (SEQ ID NO: 9)                                                             1  TTCTTGATCT                                                                               TTAGAACACT                                                                               ATGAATAGGG                                                                               AAAAAAGAAA                                                                               AAACTGTTCA                                                                               AAATAAAATG                61 TAGGAGCCGT                                                                               GCTTTTGGAA                                                                               TGCTTGAGTG                                                                               AGGAGCTCAA                                                                               CAAGTCCTCT                                                                               CCCAAGAAAG                181                                                                               CAATGATAAA                                                                               ACTTGACAAA                                                                               A                                                       98B (SEQ ID NO: 10)                                                            1  ACCCATTTCT                                                                               AACAATTTTT                                                                               ACTGTAAAAT                                                                               TTTTGGTCAA                                                                               AGTTCTAAGC                                                                               TTAATCACAT                61 CTCAAAGAAT                                                                               AGAGGCAATA                                                                               TATAGCCCAT                                                                               CTTACTAGAC                                                                               ATACAGTATT                                                                               AAACTGGACT                121                                                                               GAATATGAGG                                                                               ACAAGCTCTA                                                                               GTGGTCATTA                                                                               AACCCCTCAG                                                                               AA                                  110B (SEQ ID NO: 11)                                                           1  ACATATATTA                                                                               ACAGCATTCA                                                                               TTTGGCCAAA                                                                               ATCTACACGT                                                                               TTGTAGAATC                                                                               CTACTGTATA                61 TAAAGTGGGA                                                                               ATGTATCAAG                                                                               TATAGACTAT                                                                               GAAAGTGCAA                                                                               ATAACAAGTC                                                                               AAGGTTAGAT                121                                                                               TAACTTTTTT                                                                               TTTTTACATT                                                                               ATAAAATTAA                                                                               CTTGTTT                                       118B (SEQ ID NO: 12)                                                           1  CCAAATTTCT                                                                               CTGGAATCCA                                                                               TCCTCCCTCC                                                                               CATCACCATA                                                                               GCCTCGAGAC                                                                               GTCATTTCTG                61 TTTGACTACT                                                                               CCAGC                                                             133B (SEQ ID NO: 13)                                                           1  AACTAACCTC                                                                               CTCGGACCCC                                                                               TGCCTCACTC                                                                               ATTTACACCA                                                                               ACCACCCAAC                                                                               TATCTATAAA                61 CCTGAGCCAT                                                                               GGCCATCCCT                                                                               TATGAGCGGC                                                                               GCAGTGATTA                                                                               TAGGCTTTCG                                                                               CTCTAAGATA                121                                                                               AAAT                                                                        140B (SEQ ID NO: 14)                                                           1  ATTATTATTC                                                                               TTTTTTTATG                                                                               TTAGCTTAGC                                                                               CATGCAAAAT                                                                               TTACTGGTGA                                                                               AGCAGTTAAT                61 AAAACACACA                                                                               TCCCATTGAA                                                                               GGGTTTTGTA                                                                               CATTTCAGTC                                                                               CTTACAAATA                                                                               ACAAAGCAAT                121                                                                               GATAAACCCG                                                                               GCACGTCCTG                                                                               ATAGGAAATT                                                                               C                                             144B (SEQ ID NO: 15)                                                           1  CGTGACACAA                                                                               ACATGCATTC                                                                               GTTTTATTCA                                                                               TAAAACAGCC                                                                               TGGTTTCCTA                                                                               AAACAATACA                61 AACAGCATGT                                                                               TCATCAGCAG                                                                               GAAGCTGGCC                                                                               GTGGGCAGGG                                                                               GGGCC                               198B.sup.a (SEQ ID NO: 16)                                                     1  ATAGGTTAGA                                                                               TTCTCATTCA                                                                               CGGGACTAGT                                                                               TAGCTTTAAG                                                                               CACCCTAGAG                                                                               GACTAGGGTA                61 ATCTGACTTC                                                                               TCACTTCCTA                                                                               AGTTCCCTCT                                                                               TATATCCTCA                                                                               AGGTAGAAAT                                                                               GTCTATGTTT                121                                                                               TCTACTCCAA                                                                               TTCATAAATC                                                                               TATTCATAAG                                                                               TCTTTGGTAC                                                                               AAGTTACATG                                                                               ATAAAAAGAA                181                                                                               ATGTGATTTG                                                                               TCTTCCCTTC                                                                               TTTGCACTTT                                                                               TRAAATAAAG                                                                               TATTTATCTC                                                                               CTGTCTACAG                241                                                                               TTTAAT                                                                      212B (SEQ ID NO: 17)                                                           1  GTCCAGTATA                                                                               AAGGAAAGCG                                                                               TTAAGTCGGT                                                                               AAGCTAGAGG                                                                               ATTGTAAATA                                                                               TCTTTTATGT                61 CCTCTAGATA                                                                               AAACACCCGA                                                                               TTAACAGATG                                                                               TTAACCTTTT                                                                               ATGTTTTGAT                                                                               TTGCTTTAAA                121                                                                               AATGGCCTTC                                                                               TACACATTAG                                                                               CTCCAGCTAA                                                                               AAAGACACAT                                                                               TGAGAGCTTA                                                                               GAGGATAGTC                181                                                                               TCTGGAGC                                                                    223B.sup.b (SEQ ID NO: 18)                                                     1  GCACTTGGAA                                                                               GGGAGTTGGT                                                                               GTGCTATTTT                                                                               TGAAGCAGAT                                                                               GTGGTGATAC                                                                               TGAGATTGTC                61 TGTTCAGTTT                                                                               CCCCATTTGT                                                                               TTGTGCTTCA                                                                               AATGATCCTT                                                                               CCTACTTTGC                                                                               TTCTCTCCAC                121                                                                               CCATGACCTT                                                                               TTTCACTGTG                                                                               GCCATCAAGG                                                                               ACTTTCCTGA                                                                               CAGCTTGTGT                                                                               ACTCTTAGGC                181                                                                               TAAGAGATGT                                                                               GACTACAGCC                                                                               TGCCCCTGAC                                                                               TG                                            241B (SEQ ID NO: 19)                                                           1  TGTTAGTTTT                                                                               TAGGAAGGCC                                                                               TGTCTTCTGG                                                                               GAGTGAGGTT                                                                               TATTAGTCCA                                                                               CTTCTTGGAG                61 CTAGACGTCC                                                                               TATAGTTAGT                                                                               CACTGGGGAT                                                                               GGTGAAAGAG                                                                               GGAGAAGAGG                                                                               AAGGGCGAAG                121                                                                               GGAAGGGCTC                                                                               TTTGCTAGTA                                                                               TCTCCATTTC                                                                               TAGAAGATGG                                                                               TTTAGATGAT                                                                               AACCACAGGT                181                                                                               CTATATGAGC                                                                               ATAGTAAGGC                                                                               TGT                                                     32C.sup.b (SEQ ID NO: 20)                                                      1  CCTATTTCTG                                                                               ATCCTGACTT                                                                               TGGACAAGGC                                                                               CCTTCAGCCA                                                                               GAAGACTGAC                                                                               AAAGTCATCC                121                                                                               TCCGTCTACC                                                                               AGAGCGTGCA                                                                               CTTGTGATCC                                                                               TAAAATAAGC                                                                               TTCATCTCCG                                                                               GCTGTGCCTT                161                                                                               GGGTGGAAGG                                                                               GGCAGGATTC                                                                               TGCAGCTGCT                                                                               TTTGCATTTC                                                                               TCTTCCTAAA                                                                               TTTCATT                   34C (SEQ ID NO: 21)                                                            1  CGGAGCGTAG                                                                               GTGTGTTTAT                                                                               TCCTGTACAA                                                                               ATCATTACAA                                                                               AACCAAGTCT                                                                               GGGGCAGTCA                61 CCGCCCCCAC                                                                               CCATCACCCC                                                                               AGTGCAATGG                                                                               CTAGCTGCTG                                                                               GCCTTT                              47C (SEQ ID NO: 22)                                                            1  TTAGTTCAGT                                                                               CAAAGCAGGC                                                                               AACCCCCTTT                                                                               GGCACTGCTG                                                                               CCACTGGGGT                                                                               CATGGCGGTT                61 GTGGCAGCTG                                                                               GGGAGGTTTC                                                                               CCCAACACCC                                                                               TCCTCTGCTT                                                                               CCCTGTGTGT                                                                               CGGGGTCTCA                121                                                                               GGAGCTGACC                                                                               CAGAGTGGA                                                         65C (SEQ ID NO: 23)                                                            1  GCTGAATGTT                                                                               TAAGAGAGAT                                                                               TTTGGTCTTA                                                                               AAGGCTTCAT                                                                               CATGAAAGTG                                                                               TACATGCATA                61 TGCAAGTGTG                                                                               AATTACGTGG                                                                               TATGGATGGT                                                                               TGCTTGTTTA                                                                               TTAACTAAAG                                                                               ATGTACAGCA                121                                                                               AACTGCCCGT                                                                               TTAGAGTCCT                                                                               CTTAATATTG                                                                               ATGTCCTAAC                                                                               ACTGGGTCTG                                                                               CTTATGC                   79C (SEQ ID NO: 24)                                                            1  GGCAGTGGGA                                                                               TATGGAATCC                                                                               AGAAGGGAAA                                                                               CAAGCACTGG                                                                               ATAATTAAAA                                                                               ACAGCTGGGG                61 AGAAAACTGG                                                                               GGAAACAAAG                                                                               GATATATCCT                                                                               CATGGCTCGA                                                                               AATAAGAACA                                                                               ACGCCTGTGG                121                                                                               CATTGCCAAC                                                                               CTGGCCAGCT                                                                               TCCCCAAGAT                                                                               GTGACTCCAG                                                                               CCAGAAA                             84C (SEQ ID NO: 25)                                                            1  GCCAGGGCGG                                                                               ACCGTCTTTA                                                                               TTCCTCTCCT                                                                               GCCTCAGAGG                                                                               TCAGGAAGGA                                                                               GGTCTGGCAG                61 GACCTGCAGT                                                                               GGGCCCTAGT                                                                               CATCTGTGGC                                                                               AGCGAAGGTG                                                                               AAGGGACTCA                                                                               CCTTGTCGCC                121                                                                               CGTGCCTGAG                                                                               TAGAACTTGT                                                                               TCTGGAATTC                                                                               C                                             86C (SEQ ID NO: 26)                                                            1  AACTCTTTCA                                                                               CACTCTGGTA                                                                               TTTTTAGTTT                                                                               AACAATATAT                                                                               GTGTTGTGTC                                                                               TTGGAAATTA                61 GTTCATATCA                                                                               ATTCATATTG                                                                               AGCTGTCTCA                                                                               TTCTTTTTTT                                                                               AATGGTCATA                                                                               TACAGTAGTA                121                                                                               TTCAATTATA                                                                               AGAATATATC                                                                               CTAATACTTT                                                                               TTAAAA                                        87C (SEQ ID NO: 27)                                                            1  GGATAAGAAA                                                                               GAAGGCCTGA                                                                               GGCCTAGGGG                                                                               CCGRGGCTGG                                                                               CCTGCGTCTC                                                                               AGTCCTGGGA                61 CGCAGCAGCC                                                                               CGCACAGGTT                                                                               GAGAGGGGCA                                                                               CTTCCTCTTG                                                                               CTTAGGTTGG                                                                               TGAGGATCTG                121                                                                               GTCCTGGTTG                                                                               GCCGGTGGAG                                                                               AGCCACAAAA                                              88C (SEQ ID NO: 28)                                                            1  CTGACCTTCG                                                                               AGAGTTTGAC                                                                               CTGGAGCCGG                                                                               ATACCTACTG                                                                               CCGCTATGAC                                                                               TCGGTCAGCG                61 TGTTCAACGG                                                                               AGCCGTGAGC                                                                               GACGACTCCG                                                                               GTGGGGAAGT                                                                               TCTGCGGCGA                                                                               T                         89C (SEQ ID NO: 29)                                                            1  ATCCCTGGCT                                                                               GTGGATAGTG                                                                               CTTTTGTGTA                                                                               GCAAATGCTC                                                                               CCTCCTTAAG                                                                               GTTATAGGGC                61 TCCCTGAGTT                                                                               TGGGAGTGTG                                                                               GAAGTACTAC                                                                               TTAACTGTCT                                                                               GTCCTGCTTG                                                                               GCTGTOGTTA                121                                                                               TCGTTTTCTG                                                                               GTGATGTTGT                                                                               GCTAACAATA                                                                               AGAATAC                                       101C (SEQ ID NO: 30)                                                           1  GGCTGGGCAT                                                                               CCCTCTCCTC                                                                               CTCCATCCCC                                                                               ATACATCACC                                                                               AGGTCTAATG                                                                               TTTACAAACG                61 GTGCCAGCCC                                                                               GGCTCTGAAG                                                                               CCAAGGGCCG                                                                               TCCGTGCCAC                                                                               GGTGGCTGTG                                                                               AGTATTCCTC                121                                                                               CGTTAGCTTT                                                                               CCCATAAGGT                                                                               TGGAGTATCT                                                                               GC                                            112C (SEQ ID NO: 31)                                                           1  CCAACTCCTA                                                                               CCGCGATACA                                                                               GACCCACAGA                                                                               GTGCCATCCC                                                                               TGAGAGACCA                                                                               GACCGCTCCC                161                                                                               CAATACTCTC                                                                               CTAAAATAAA                                                                               CATGAAGCAC                                              114C (SEQ ID NO: 32)                                                           1  CATGGATGAA                                                                               TGTCTCATGG                                                                               TGGGAAGGAA                                                                               CATGGTACAT                                                                               TTC                                 __________________________________________________________________________      .sup.a Repeated 3 times                                                        .sup.b Repeated 2 times                                                  

Sequence analysis of the OC⁺ stromal cell⁻ cloned DNA sequences revealed, in addition to the novel sequences, a number of previously-described genes. The known genes identified (including type 5 acid phosphatase, gelatinase B, cystatin C (13 clones), Alu repeat sequences (11 clones), creatnine kinase (6 clones) and others) are summarized in Table II. In situ hybridization (described below) directly demonstrated that gelatinase B mRNA is expressed in multinucleated osteoclasts and not in stromal cells. Although gelatinase B is a well-characterized protease, its expression at high levels in osteoclasts has not been previously described. The expression in osteoclasts of cystatin C, a cysteine protease inhibitor, is also unexpected. This finding has not yet been confirmed by in situ hybridization. Taken together, these results demonstrate that most of these identified genes are osteoclast-expressed, thereby confirming the effectiveness of the differential screening strategy for identifying DNA encoding osteoclast-specific or -related gene products. Therefore, novel genes identified by this method have a high probability of being OC-specific or related.

In addition, a minority of the genes identified by this screen are probably not expressed by OCs (Table II). For example, type III collagen (6 clones), collagen type I (1 clone), dermatansulfate (1 clone), and type VI collagen (1 clone) are more likely to originate from the stromal cells or from osteoblastic cells which are present in the tumor. These cDNA sequences survive the differential screening process either because the cells which produce them in the tumor in vivo die out during the stromal cell propagation phase, or because they stop producing their product in vitro. These clones do not constitute more than 5-10% of the all sequences selected by differential hybridization.

                  TABLE II                                                         ______________________________________                                         SEQUENCE ANALYSIS OF CLONES ENCODING KNOWN                                     SEQUENCES FROM AN OSTEOCLASTOMA cDNA                                           LIBRARY                                                                        Clones with Sequence Homology                                                                               25 total                                          to Collagenase Type IV                                                         Clones with Sequence Homology to                                                                            14 total                                          Type 5 Tartrate Resistant Acid Phosphatase                                     Clones with Sequence Homology to                                                                            13 total                                          Cystatin C:                                                                    Clones with Sequence Homology to                                                                            11 total                                          Alu-repeat Sequences                                                           Clones with Sequence Homology to                                                                             6 total                                          Creatnine Kinase                                                               Clones with Sequence Homology to                                                                             6 total                                          Type III Collagen                                                              Clones with Sequence Homology to                                                                             5 total                                          MHC Class I γ Invariant Chain                                            Clones with Sequence Homology to                                                                             3 total                                          MHC Class II β Chain                                                      One or Two Clone(s) with Sequence Homology to Each                                                          10 total                                          of the Following:                                                              αI collagen type I                                                       γ interferon inducible protein                                           osteopontin                                                                    Human chondroitin/dermatansulfate                                              α globin                                                                 β glucosidase/sphingolipid activator                                      Human CAPL protein (Ca binding)                                                Human EST 01024                                                                Type VI collagen                                                               Human EST 00553                                                                ______________________________________                                    

Example 5--In situ Hybridiation of OC-Expressed Genes

In situ hybridization was performed using probes derived from novel cloned sequences in order to determine whether the novel putative OC-specific or -related genes are differentially expressed in osteoclasts (and not expressed in the stromal cells) of human giant cell tumors. Initially, in situ hybridization was performed using antisense (positive) and sense (negative control) cRNA probes against human type IV collagenase/gelatinase B labelled with ³⁵ S-UTP.

A thin section of human giant cell tumor reacted with the antisense probe resulted in intense labelling of all OCs, as indicated by the deposition of silver grains over these cells, but failed to label the stromal cell elements. In contrast, only minimal background labelling was observed with the sense (negative control) probe. This result confirmed that gelatinase B is expressed in human OCs.

In situ hybridization was then carried out using cRNA probes derived from 11/32 novel genes, labelled with digoxigenin UTP according to known methods.

The results of this analysis are summarized in Table III. Clones 28B, 118B, 140B, 198B, and 212B all gave positive reactions with OCs in frozen sections of a giant cell tumor, as did the positive control gelatinase B. These novel clones therefore are expressed in OCs and fulfill all criteria for OC-relatedness. 198B is repeated three times, indicating relatively high expression. Clones 4B, 37B, 88C and 98B produced positive reactions with the tumor tissue; however the signal was not well-localized to OCs. These clones are therefore not likely to be useful and are eliminated from further consideration. Clones 86B and 87B failed to give a positive reaction with any cell type, possibly indicating very low level expression. This group of clones could still be useful but may be difficult to study further. The results of this analysis show that 5/11 novel genes are expressed in OCs, indicating that ˜50% of novel sequences likely to be OC-related.

To generate probes for the in situ hybridizations, cDNA derived from novel cloned osteoclast-specific or -related cDNA was subcloned into a BlueScript II SK(-) vector. The orientation of cloned inserts was determined by restriction analysis of subclones. The T7 and T3 promoters in the BlueScriptII vector was used to generate ³⁵ S-labelled (³⁵ S-UTP 850 Ci/mmol, Amersham, Arlington Heights, Ill.), or UTP digoxygenin labelled cRNA probes.

                  TABLE III                                                        ______________________________________                                         In Situ HYBRIDIZATION USING PROBES                                             DERIVED FROM NOVEL SEQUENCES                                                                Reactivity with:                                                  Clone          Osteoclasts                                                                              Stromal Cells                                         ______________________________________                                         4B             +         +                                                     28B*           +         -                                                     37B            +         +                                                     86B            -         -                                                     87B            -         -                                                     88C            +         +                                                     98B            +         +                                                     118B*          +         -                                                     140B*          +         -                                                     198B*          +         -                                                     212B*          +         -                                                     Gelatinase B*  +         -                                                     ______________________________________                                          *OC-expressed, as indicated by reactivity with antisense probe and lack o      reactivity with sense probe on OCs only.                                 

In situ hybridization was carried out on 7 micron cryostat sections of a human osteoclastoma as described previously (Chang, L.-C. et al. Cancer Res. 49:6700 (1989)). Briefly, tissue was fixed in 4% paraformaldehyde and embedded in OCT (Miles Inc., Kankakee, Ill.). The sections were rehydrated, postfixed in 4% paraformaldehyde, washed, and pretreated with 10 mM DTT, 10 mM iodoacetamide, 10 mM N-ethylmaleimide and 0.1 triethanolamine-HCL. Prehybridization was done with 50% deionized formamide, 10 mM Tris-HCl, pH 7.0, 1× Denhardt's, 500 mg/ml tRNA, 80 mg/ml salmon sperm DNA, 0.3M NaCl, mM EDTA, and 100 mM DTT at 45° C. for 2 hours. Fresh hybridization solution containing 10% dextran sulfate and 1.5 ng/ml ³⁵ S-labelled or digoxygenin labelled RNA probe was applied after heat denaturation. Sections were coverslipped and then incubated in a moistened chamber at 45°-50° C. overnight. Hybridized sections were washed four times with 50% formamide, 2× SSC, containing 10 mM DTT and 0.5% Triton X-100 at 45° C. Sections were treated with RNase A and RNase T1 to digest single-stranded RNA, washed four times in 2× SSC/10 mM DTT.

In order to detect ³⁵ S-labelling by autoradiography, slides were dehydrated, dried, and coated with Kodak NTB-2 emulsion. The duplicate slides were split, and each set was placed in a black box with desiccant, sealed, and incubated at 4° C. for 2 days. The slides were developed (4 minutes) and fixed (5 minutes) using Kodak developer D19 and Kodak fixer. Hematoxylin and eosin were used as counter-stains.

In order to detect digoxygenin-labelled probes, a Nucleic Acid Detection Kit (Boehringer-Mannheim, Cat. #1175041) was used. Slides were washed in Buffer 1 consisting of 100 mM Tris/150 mM NaCl, pH7.5, for 1 minute. 100 μl Buffer 2 was added (made by adding 2 mg/ml blocking reagent as provided by the manufacturer) in Buffer 1 to each slide. The slides were placed on a shaker and gently swirled at 20° C.

Antibody solutions were diluted 1:100 with Buffer 2 (as provided by the manufacturer). 100 μl of diluted antibody solution was applied to the slides and the slides were then incubated in a chamber for 1 hour at room temperature. The slides were monitored to avoid drying. After incubation with antibody solution, slides were washed in Buffer 1 for 10 minutes, then washed in Buffer 3 containing 2 mM levamisole for 2 minutes.

After washing, 100 μl color solution was added to the slides. Color solution consisted of nitroblue/tetrazolium salt (NBT) (1:225 dilution) 4.5 μl, 5-bromo-4-chloro-3-indolyl phosphate (1:285 dilution) 3.5 μl, levamisole 0.2 mg in Buffer 3 (as provided by the manufacturer) in a total volume of 1 ml. Color solution was prepared immediately before use.

After adding the color solution, the slides were placed in a dark, humidified chamber at 20° C. for 2-5 hours and monitored for color development. The color reaction was stopped by rinsing slides in TE Buffer.

The slides were stained for 60 seconds in 0.25% methyl green, washed with tap water, then mounted with water-based Permount (Fisher).

Example 6--Immunohistochemistry

Immunohistochemical staining was performed on frozen and paraffin embedded tissues as well as on cytospin preparations (see Table IV). The following antibodies were used: polyclonal rabbit anti-human gelatinase antibodies; Ab110 for gelatinase B; monoclonal mouse anti-human CD68 antibody (clone KP1) (DAKO, Denmark); Mol (anti-CD11b) and Mo2 (anti-CD14) derived from ATCC cell lines HB CRL 8026 and TIB 228/HB44. The anti-human gelatinase B antibody Ab110 was raised against a synthetic peptide with the amino acid sequence EALMYPMYRFTEGPPLHK (SEQ ID NO: 34), which is specific for human gelatinase B (Corcoran, M. L. et al. J. Biol. Chem, 267:515 (1992)).

Detection of the immunohistochemical staining was achieved by using a goat anti-rabbit glucose oxidase kit (Vector Laboratories, Burlingame Calif.) according to the manufacturer's directions. Briefly, the sections were rehydrated and pretested with either acetone or 0.1% trypsin. Normal goat serum was used to block nonspecific binding. Incubation with the primary antibody for 2 hours or overnight (Abl10:1/500 dilution) was followed by either a glucose oxidase labeled secondary anti-rabbit serum, or, in the case of the mouse monoclonal antibodies, were reacted with purified rabbit anti-mouse Ig before incubation with the secondary antibody.

Paraffin embedded and frozen sections from osteoclastomas (GCT) were reacted with a rabbit antiserum against gelatinase B (antibody 110) (Corcoran, M. L. et al. J. Biol. chem. 267:515 (1992)), followed by color development with glucose oxidase linked reagents. The osteoclasts of a giant cell tumor were uniformly strongly positive for gelatinase B, whereas the stromal cells were unreactive. Control sections reacted with rabbit preimmune serum were negative. Identical findings were obtained for all 8 long bone giant cell tumors tested (Table IV). The osteoclasts present in three out of four central giant cell granulomas (GCG) of the mandible were also positive for gelatinase B expression. These neoplasms are similar but not identical to the long bone giant cell tumors, apart from their location in the jaws (Shafer, W. G. et al., Textbook of Oral Pathology, W. B. Saunders Company, Philadelphia, pp. 144-149 (1983)). In contrast, the multinucleated cells from a peripheral giant cell tumor, which is a generally non-resorptive tumor of oral soft tissue, were unreactive with antibody (Shafer, W. G. et. al., Textbook of Oral Pathology, W. B. Saunders Company, Philadelphia, pp. 144-149 (1983)).

Antibody 110 was also utilized to assess the presence of gelatinase B in normal bone (n=3) and in Paget's disease, in which there is elevated bone remodeling and increased osteoclastic activity. Strong staining for gelatinase B was observed in osteoclasts both in normal bone (mandible of a 2 year old), and in Paget's disease. Staining was again absent in controls incubated with preimmune serum. Osteoblasts did not stain in any of the tissue sections, indicating that gelatinase B expression is limited to osteoclasts in bone. Finally, peripheral blood monocytes were also reactive with antibody 110 (Table IV).

                  TABLE IV                                                         ______________________________________                                         DISTRIBUTION OF GELATINASE B IN VARIOUS                                        TISSUES                                                                                       Antibodies tested                                                              Ab 110                                                          Samples        gelatinase B                                                    ______________________________________                                         GCT frozen                                                                     (n = 2)                                                                        giant cells    +                                                               stromal cells  -                                                               GCT paraffin                                                                   (n = 6)                                                                        giant cells    +                                                               stromal cells  -                                                               central GCG                                                                    (n = 4)                                                                        giant cells      +(3/4)                                                        stromal cells  -                                                               peripheral GCT                                                                 (n - 4)                                                                        giant cells    -                                                               stromal cells  -                                                               Paget's disease                                                                (n = 1)                                                                        osteoclasts    +                                                               osteoblasts    -                                                               normal bone                                                                    (n = 3)                                                                        osteoclasts    +                                                               osteoblasts    -                                                               monocytes      +                                                               (cytospin)                                                                     ______________________________________                                          Distribution of gelatinase B in multinucleated giant cells, osteoclasts,       osteoblasts and stromal cells in various tissues. In general, paraffin         embedded tissues were used for these experiments; exceptions are               indicated.                                                               

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 34                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 170 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        GCAAATATCTAAGTTTATTGCTTGGATTTCTAGTGAGAGCTGTTGAATTTGGTGATGTCA60                 AATGTTTCTAGGGTTTTTTTAGTTTGTTTTTATTGAAAAATTTAATTATTTATGCTATAG120                GTGATATTCTCTTTGAATAAACCTATAATAGAAAATAGCAGCAGACAACA170                          (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 63 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        GTGTCAACCTGCATATCCTAAAAATGTCAAAATGCTGCATCTGGTTAATGTCGGGGTAGG60                 GGG63                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 163 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        CTTCCCTCTCTTGCTTCCCTTTCCCAAGCAGAGGTGCTCACTCCATGGCCACCGCCACCA60                 CAGGCCCACAGGGAGTACTGCCAGACTACTGCTGATGTTCTCTTAAGGCCCAGGGAGTCT120                CAACCAGCTGGTGGTGAATGCTGCCTGGCACGGGACCCCCCCC163                                 (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 173 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        TTTTATTTGTAAATATATGTATTACATCCCTAGAAAAAGAATCCCAGGATTTTCCCTCCT60                 GTGTGTTTTCGTCTTGCTTCTTCATGGTCCATGATGCCAGCTGAGGTTGTCAGTACAATG120                AAACCAAACTGGCGGGATGGAAGCAGATTATTCTGCCATTTTTCCAGGTCTTT173                       (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 197 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        GGCTGGACATGGGTGCCCTCCACGTCCCTCATATCCCCAGGCACACTCTGGCCTCAGGTT60                 TTGCCCTGGCCATGTCATCTACCTGGAGTGGGCCCTCCCCTTCTTCAGCCTTGAATCAAA120                AGCCACTTTGTTAGGCGAGGATTTCCCAGACCACTCATCACATTAAAAAATATTTTGAAA180                ACAAAAAAAAAAAAAAA197                                                           (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 132 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        TTGACAAAGCTGTTTATTTCCACCAATAAATAGTATATGGTGATTGGGGTTTCTATTTAT60                 AAGAGTAGTGGCTATTATATGGGGTATCATGTTGATGCTCATAAATAGTTCATATCTACT120                TAATTTGCCTTC132                                                                (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 75 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        GAAGAGAGTTGTATGTACAACCCCAACAGGCAAGGCAGCTAAATGCAGAGGGTACAGAGA60                 GATCCCGAGGGAATT75                                                              (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 151 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        GGATGGAAACATGTAGAAGTCCAGAGAAAAACAATTTTAAAAAAAGGTGGAAAAGTTACG60                 GCAAACCTGAGATTTCAGCATAAAATCTTTAGTTAGAAGTGAGAGAAAGAAGAGGGAGGC120                TGGTTGCTGTTGCACGTATCAATAGGTTATC151                                             (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 141 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        TTCTTGATCTTTAGAACACTATGAATAGGGAAAAAAGAAAAAACTGTTCAAAATAAAATG60                 TAGGAGCCGTGCTTTTGGAATGCTTGAGTGAGGAGCTCAACAAGTCCTCTCCCAAGAAAG120                CAATGATAAAACTTGACAAAA141                                                       (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 162 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       ACCCATTTCTAACAATTTTTACTGTAAAATTTTTGGTCAAAGTTCTAAGCTTAATCACAT60                 CTCAAAGAATAGAGGCAATATATAGCCCATCTTACTAGACATACAGTATTAAACTGGACT120                GAATATGAGGACAAGCTCTAGTGGTCATTAAACCCCTCAGAA162                                  (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 157 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       ACATATATTAACAGCATTCATTTGGCCAAAATCTACACGTTTGTAGAATCCTACTGTATA60                 TAAAGTGGGAATGTATCAAGTATAGACTATGAAAGTGCAAATAACAAGTCAAGGTTAGAT120                TAACTTTTTTTTTTTACATTATAAAATTAACTTGTTT157                                       (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 75 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       CCAAATTTCTCTGGAATCCATCCTCCCTCCCATCACCATAGCCTCGAGACGTCATTTCTG60                 TTTGACTACTCCAGC75                                                              (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 124 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                       AACTAACCTCCTCGGACCCCTGCCTCACTCATTTACACCAACCACCCAACTATCTATAAA60                 CCTGAGCCATGGCCATCCCTTATGAGCGGCGCAGTGATTATAGGCTTTCGCTCTAAGATA120                AAAT124                                                                        (2) INFORMATION FOR SEQ ID NO:14:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 151 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                       ATTATTATTCTTTTTTTATGTTAGCTTAGCCATGCAAAATTTACTGGTGAAGCAGTTAAT60                 AAAACACACATCCCATTGAAGGGTTTTGTACATTTCAGTCCTTACAAATAACAAAGCAAT120                GATAAACCCGGCACGTCCTGATAGGAAATTC151                                             (2) INFORMATION FOR SEQ ID NO:15:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 105 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       CGTGACACAAACATGCATTCGTTTTATTCATAAAACAGCCTGGTTTCCTAAAACAATACA60                 AACAGCATGTTCATCAGCAGGAAGCTGGCCGTGGGCAGGGGGGCC105                               (2) INFORMATION FOR SEQ ID NO:16:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 246 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       ATAGGTTAGATTCTCATTCACGGGACTAGTTAGCTTTAAGCACCCTAGAGGACTAGGGTA60                 ATCTGACTTCTCACTTCCTAAGTTCCCTCTTATATCCTCAAGGTAGAAATGTCTATGTTT120                TCTACTCCAATTCATAAATCTATTCATAAGTCTTTGGTACAAGTTACATGATAAAAAGAA180                ATGTGATTTGTCTTCCCTTCTTTGCACTTTTGAAATAAAGTATTTATCTCCTGTCTACAG240                TTTAAT246                                                                      (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 188 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       GTCCAGTATAAAGGAAAGCGTTAAGTCGGTAAGCTAGAGGATTGTAAATATCTTTTATGT60                 CCTCTAGATAAAACACCCGATTAACAGATGTTAACCTTTTATGTTTTGATTTGCTTTAAA120                AATGGCCTTCTACACATTAGCTCCAGCTAAAAAGACACATTGAGAGCTTAGAGGATAGTC180                TCTGGAGC188                                                                    (2) INFORMATION FOR SEQ ID NO:18:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 212 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                       GCACTTGGAAGGGAGTTGGTGTGCTATTTTTGAAGCAGATGTGGTGATACTGAGATTGTC60                 TGTTCAGTTTCCCCATTTGTTTGTGCTTCAAATGATCCTTCCTACTTTGCTTCTCTCCAC120                CCATGACCTTTTTCACTGTGGCCATCAAGGACTTTCCTGACAGCTTGTGTACTCTTAGGC180                TAAGAGATGTGACTACAGCCTGCCCCTGACTG212                                            (2) INFORMATION FOR SEQ ID NO:19:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 203 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                       TGTTAGTTTTTAGGAAGGCCTGTCTTCTGGGAGTGAGGTTTATTAGTCCACTTCTTGGAG60                 CTAGACGTCCTATAGTTAGTCACTGGGGATGGTGAAAGAGGGAGAAGAGGAAGGGCGAAG120                GGAAGGGCTCTTTGCTAGTATCTCCATTTCTAGAAGATGGTTTAGATGATAACCACAGGT180                CTATATGAGCATAGTAAGGCTGT203                                                     (2) INFORMATION FOR SEQ ID NO:20:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 177 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                       CCTATTTCTGATCCTGACTTTGGACAAGGCCCTTCAGCCAGAAGACTGACAAAGTCATCC60                 TCCGTCTACCAGAGCGTGCACTTGTGATCCTAAAATAAGCTTCATCTCCGGCTGTGCCTT120                GGGTGGAAGGGGCAGGATTCTGCAGCTGCTTTTGCATTTCTCTTCCTAAATTTCATT177                   (2) INFORMATION FOR SEQ ID NO:21:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 106 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       CGGAGCGTAGGTGTGTTTATTCCTGTACAAATCATTACAAAACCAAGTCTGGGGCAGTCA60                 CCGCCCCCACCCATCACCCCAGTGCAATGGCTAGCTGCTGGCCTTT106                              (2) INFORMATION FOR SEQ ID NO:22:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 139 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                       TTAGTTCAGTCAAAGCAGGCAACCCCCTTTGGCACTGCTGCCACTGGGGTCATGGCGGTT60                 GTGGCAGCTGGGGAGGTTTCCCCAACACCCTCCTCTGCTTCCCTGTGTGTCGGGGTCTCA120                GGAGCTGACCCAGAGTGGA139                                                         (2) INFORMATION FOR SEQ ID NO:23:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 177 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                       GCTGAATGTTTAAGAGAGATTTTGGTCTTAAAGGCTTCATCATGAAAGTGTACATGCATA60                 TGCAAGTGTGAATTACGTGGTATGGATGGTTGCTTGTTTATTAACTAAAGATGTACAGCA120                AACTGCCCGTTTAGAGTCCTCTTAATATTGATGTCCTAACACTGGGTCTGCTTATGC177                   (2) INFORMATION FOR SEQ ID NO:24:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 167 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                       GGCAGTGGGATATGGAATCCAGAAGGGAAACAAGCACTGGATAATTAAAAACAGCTGGGG60                 AGAAAACTGGGGAAACAAAGGATATATCCTCATGGCTCGAAATAAGAACAACGCCTGTGG120                CATTGCCAACCTGGCCAGCTTCCCCAAGATGTGACTCCAGCCAGAAA167                             (2) INFORMATION FOR SEQ ID NO:25:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 151 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                       GCCAGGGCGGACCGTCTTTATTCCTCTCCTGCCTCAGAGGTCAGGAAGGAGGTCTGGCAG60                 GACCTGCAGTGGGCCCTAGTCATCTGTGGCAGCGAAGGTGAAGGGACTCACCTTGTCGCC120                CGTGCCTGAGTAGAACTTGTTCTGGAATTCC151                                             (2) INFORMATION FOR SEQ ID NO:26:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 156 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                       AACTCTTTCACACTCTGGTATTTTTAGTTTAACAATATATGTGTTGTGTCTTGGAAATTA60                 GTTCATATCAATTCATATTGAGCTGTCTCATTCTTTTTTTAATGGTCATATACAGTAGTA120                TTCAATTATAAGAATATATCCTAATACTTTTTAAAA156                                        (2) INFORMATION FOR SEQ ID NO:27:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 150 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                       GGATAAGAAAGAAGGCCTGAGGGCTAGGGGCCGGGGCTGGCCTGCGTCTCAGTCCTGGGA60                 CGCAGCAGCCCGCACAGGTTGAGAGGGGCACTTCCTCTTGCTTAGGTTGGTGAGGATCTG120                GTCCTGGTTGGCCGGTGGAGAGCCACAAAA150                                              (2) INFORMATION FOR SEQ ID NO:28:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 212 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                       GCACTTGGAAGGGAGTTGGTGTGCTATTTTTGAAGCAGATGTGGTGATACTGAGATTGTC60                 TGTTCAGTTTCCCCATTTGTTTGTGCTTCAAATGATCCTTCCTACTTTGCTTCTCTCCAC120                CCATGACCTTTTTCACTGTGGCCATCAAGGACTTTCCTGACAGCTTGTGTACTCTTAGGC180                TAAGAGATGTGACTACAGCCTGCCCCTGACTG212                                            (2) INFORMATION FOR SEQ ID NO:29:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 157 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                       ATCCCTGGCTGTGGATAGTGCTTTTGTGTAGCAAATGCTCCCTCCTTAAGGTTATAGGGC60                 TCCCTGAGTTTGGGAGTGTGGAAGTACTACTTAACTGTCTGTCCTGCTTGGCTGTCGTTA120                TCGTTTTCTGGTGATGTTGTGCTAACAATAAGAATAC157                                       (2) INFORMATION FOR SEQ ID NO:30:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 152 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                       GGCTGGGCATCCCTCTCCTCCTCCATCCCCATACATCACCAGGTCTAATGTTTACAAACG60                 GTGCCAGCCCGGCTCTGAAGCCAAGGGCCGTCCGTGCCACGGTGGCTGTGAGTATTCCTC120                CGTTAGCTTTCCCATAAGGTTGGAGTATCTGC152                                            (2) INFORMATION FOR SEQ ID NO:31:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 90 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                       CCAACTCCTACCGCGATACAGACCCACAGAGTGCCATCCCTGAGAGACCAGACCGCTCCC60                 CAATACTCTCCTAAAATAAACATGAAGCAC90                                               (2) INFORMATION FOR SEQ ID NO:32:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 43 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                       CATGGATGAATGTCTCATGGTGGGAAGGAACATGGTACATTTC43                                  (2) INFORMATION FOR SEQ ID NO:33:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2333 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                       AGACACCTCTGCCCTCACCATGAGCCTCTGGCAGCCCCTGGTCCTGGTGCTCCTGGTGCT60                 GGGCTGCTGCTTTGCTGCCCCCAGACAGCGCCAGTCCACCCTTGTGCTCTTCCCTGGAGA120                CCTGAGAACCAATCTCACCGACAGGCAGCTGGCAGAGGAATACCTGTACCGCTATGGTTA180                CACTCGGGTGGCAGAGATGCGTGGAGAGTCGAAATCTCTGGGGCCTGCGCTGCTGCTTCT240                CCAGAAGCAACTGTCCCTGCCCGAGACCGGTGAGCTGGATAGCGCCACGCTGAAGGCCAT300                GCGAACCCCACGGTGCGGGGTCCCAGACCTGGGCAGATTCCAAACCTTTGAGGGCGACCT360                CAAGTGGCACCACCACAACATCACCTATTGGATCCAAAACTACTCGGAAGACTTGCCGCG420                GGCGGTGATTGACGACGCCTTTGCCCGCGCCTTCGCACTGTGGAGCGCGGTGACGCCGCT480                CACCTTCACTCGCGTGTACAGCCGGGACGCAGACATCGTCATCCAGTTTGGTGTCGCGGA540                GCACGGAGACGGGTATCCCTTCGACGGGAAGGACGGGCTCCTGGCACACGCCTTTCCTCC600                TGGCCCCGGCATTCAGGGAGACGCCCATTTCGACGATGACGAGTTGTGGTCCCTGGGCAA660                GGGCGTCGTGGTTCCAACTCGGTTTGGAAACGCAGATGGCGCGGCCTGCCACTTCCCCTT720                CATCTTCGAGGGCCGCTCCTACTCTGCCTGCACCACCGACGGTCGCTCCGACGGGTTGCC780                CTGGTGCAGTACCACGGCCAACTACGACACCGACGACCGGTTTGGCTTCTGCCCCAGCGA840                GAGACTCTACACCCGGGACGGCAATGCTGATGGGAAACCCTGCCAGTTTCCATTCATCTT900                CCAAGGCCAATCCTACTCCGCCTGCACCACGGACGGTCGCTCCGACGGCTACCGCTGGTG960                CGCCACCACCGCCAACTACGACCGGGACAAGCTCTTCGGCTTCTGCCCGACCCGAGCTGA1020               CTCGACGGTGATGGGGGGCAACTCGGCGGGGGAGCTGTGCGTCTTCCCCTTCACTTTCCT1080               GGGTAAGGAGTACTCGACCTGTACCAGCGAGGGCCGCGGAGATGGGCGCCTCTGGTGCGC1140               TACCACCTCGAACTTTGACAGCGACAAGAAGTGGGGCTTCTGCCCGGACCAAGGATACAG1200               TTTGTTCCTCGTGGCGGCGCATGAGTTCGGCCACGCGCTGGGCTTAGATCATTCCTCAGT1260               GCCGGAGGCGCTCATGTACCCTATGTACCGCTTCACTGAGGGGCCCCCCTTGCATAAGGA1320               CGACGTGAATGGCATCCGGCACCTCTATGGTCCTCGCCCTGAACCTGAGCCACGGCCTCC1380               AACCACCACCACACCGCAGCCCACGGCTCCCCCGACGGTCTGCCCCACCGGACCCCCCAC1440               TGTCCACCCCTCAGAGCGCCCCACAGCTGGCCCCACAGGTCCCCCCTCAGCTGGCCCCAC1500               AGGTCCCCCCACTGCTGGCCCTTCTACGGCCACTACTGTGCCTTTGAGTCCGGTGGACGA1560               TGCCTGCAACGTGAACATCTTCGACGCCATCGCGGAGATTGGGAACCAGCTGTATTTGTT1620               CAAGGATGGGAAGTACTGGCGATTCTCTGAGGGCAGGGGGAGCCGGCCGCAGGGCCCCTT1680               CCTTATCGCCGACAAGTGGCCCGCGCTGCCCCGCAAGCTGGACTCGGTCTTTGAGGAGCC1740               GCTCTCCAAGAAGCTTTTCTTCTTCTCTGGGCGCCAGGTGTGGGTGTACACAGGCGCGTC1800               GGTGCTGGGCCCGAGGCGTCTGGACAAGCTGGGCCTGGGAGCCGACGTGGCCCAGGTGAC1860               CGGGGCCCTCCGGAGTGGCAGGGGGAAGATGCTGCTGTTCAGCGGGCGGCGCCTCTGGAG1920               GTTCGACGTGAAGGCGCAGATGGTGGATCCCCGGAGCGCCAGCGAGGTGGACCGGATGTT1980               CCCCGGGGTGCCTTTGGACACGCACGACGTCTTCCAGTACCGAGAGAAAGCCTATTTCTG2040               CCAGGACCGCTTCTACTGGCGCGTGAGTTCCCGGAGTGAGTTGAACCAGGTGGACCAAGT2100               GGGCTACGTGACCTATGACATCCTGCAGTGCCCTGAGGACTAGGGCTCCCGTCCTGCTTT2160               GCAGTGCCATGTAAATCCCCACTGGGACCAACCCTGGGGAAGGAGCCAGTTTGCCGGATA2220               CAAACTGGTATTCTGTTCTGGAGGAAAGGGAGGAGTGGAGGTGGGCTGGGCCCTCTCTTC2280               TCACCTTTGTTTTTTGTTGGAGTGTTTCTAATAAACTTGGATTCTCTAACCTTT2334                     (2) INFORMATION FOR SEQ ID NO:34:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                       GluAlaLeuMetTyrProMetTyrArgPheThrGluGlyProProLeu                               151015                                                                         HisLys                                                                         __________________________________________________________________________ 

We claim:
 1. An isolated osteoclast-specific or -related DNA sequence, or its complementary sequence, the DNA sequence comprising a nucleic acid sequence selected from the group consisting of:a) DNA sequences set forth in the group consisting of SEQ ID NOS. 12, 14, 16 and 17, or their complementary strands; and b) DNA sequences which hybridize under standard conditions to the DNA sequences defined in a).
 2. A DNA construct capable of replicating, in a host cell, osteoclast-specific or -related DNA, said construct comprising:a) a DNA sequence of claim 1; and b) sequences, in addition to said DNA sequence, necessary for transforming or transfecting a host cell, and for replicating, in a host cell, said DNA sequence.
 3. A DNA construct capable or replicating and expressing, in a host cell, osteoclast-specific or -related DNA, said construct comprising:a) a DNA sequence of claim 2; and b) sequences, in addition to said DNA sequence, necessary for transforming or transfecting a host cell, and for replicating and expressing, in a host cell, said DNA sequence.
 4. A cell stably transformed or transfected with a DNA construct according to claim
 3. 5. A cell stably transformed or transfected with a DNA construct according to claim
 4. 